Nanopore flow cells and methods of fabrication

ABSTRACT

Nanopore flow cells and methods of manufacturing thereof are provided herein. In one embodiment a method of forming a flow cell includes forming a multilayer stack on a first substrate, e.g., a monocrystalline silicon substrate, before transferring the multilayer stack to a second substrate, e.g., a glass substrate. Here, the multilayer stack features a membrane layer, having a first opening formed therethrough, where the membrane layer is disposed on the first substrate, and a material layer is disposed on the membrane layer. The method further includes patterning the second substrate to form a second opening therein and bonding the patterned surface of the second substrate to a surface of the multilayer stack. The method further includes thinning the first substrate and thinning the second substrate. Here, the second substrate is thinned to where the second opening is disposed therethrough. The method further includes removing the thinned first substrate and at least portions of the material layer to expose opposite surfaces of the membrane layer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Divisional of U.S. application Ser. No.16/573,540, filed on Sep. 17, 2019, which issues on Feb. 15, 2022 asU.S. Pat. No. 11,249,067, which claims the benefit of U.S. ProvisionalApplication 62/752,045 filed on Oct. 29, 2018, each of which areincorporated by reference in their entirety.

BACKGROUND Field

Embodiments herein relate to flow cells to be used with solid-statenanopore sensors and methods of manufacturing thereof.

Description of the Related Art

Solid-state nanopore sensors have emerged as a low-cost, highly mobile,and rapid processing biopolymer, e.g., DNA or RNA, sequencingtechnology. Solid-state nanopore sequencing of a biopolymer strandcomprises translocating the biopolymer strand through a nanoscale sizedopening having a diameter between about 0.1 nm and about 100 nm, i.e., ananopore. Typically, the nanopore is disposed through a membrane layerwhich separates two conductive fluid reservoirs. The biopolymer strandto be sequenced, e.g., a characteristically negatively charged DNA orRNA strand, is introduced into one of the two conductive fluidreservoirs and is then drawn through the nanopore by providing anelectric potential therebetween. As the biopolymer strand travelsthrough the nanopore the different monomer units thereof, e.g., proteinbases of a DNA or RNA strand, occlude different percentages of thenanopore thus changing the ionic current flow therethrough. Theresulting current signal pattern can be used to determine the sequenceof monomer units in the biopolymer strand, such as the sequence ofproteins in a DNA or RNA strand.

Often, the membrane layer and the nanopore disposed therethrough arefabricated on a monocrystalline silicon substrate which togethertherewith forms a nanopore flow cell. The monocrystalline siliconsubstrate is typically the same or similar to substrates used in themanufacturing of semiconductor devices. Using the same or similarsubstrate to those used in the manufacture of semiconductor devicesfacilitates fabrication of the nanopore flow cell using commerciallyavailable semiconductor device manufacturing equipment and methods.

Typically, a membrane layer is deposited onto a front side surface of asilicon substrate and the nanopore opening is formed through themembrane layer, but not through the silicon substrate, using aphotolithography patterning and etch processing sequence. A surface ofthe membrane layer disposed proximate to the silicon substrate is thenexposed by etching an opening into a backside surface of the siliconsubstrate. Typically, the opening in the backside surface of the siliconsubstrate is formed by exposing the backside surface of the substrate toa wet or aqueous silicon etchant, such as KOH, through a patterned maskdisposed thereon. A typical silicon substrate will need to be exposed tothe silicon etchant for between 9 and 13 hours to anisotropically etchthrough the thickness thereof. This long etch time undesirably increasesthe cycle time, and thus the cost, of forming the nanopore flow cell.Further, charges accumulated in the monocrystalline substrate used tosupport the membrane layer during high frequency nucleotide detection ina conventional nanopore flow cell undesirably increase background noisein the current signal. This undesirable background noise reduces thedetection resolution of the nanopore sensor or flow cell.

Accordingly, what is needed in the art are improved methods of forming ananopore flow cell for use in a solid-state nanopore sensor and improvednanopore flow cells formed therefrom.

SUMMARY

Embodiments of the present disclosure provide devices, e.g., nanoporeflow cells, which may be used in a solid-state nanopore sensor, andmethods of manufacturing thereof.

In one embodiment a method of forming a flow cell includes forming amultilayer stack on a first substrate, e.g., a monocrystalline siliconsubstrate, before transferring the multilayer stack to a secondsubstrate, e.g., a glass substrate. Here, the multilayer stack featuresa membrane layer, having a first opening formed therethrough, where themembrane layer is disposed on the first substrate, and a material layeris disposed on the membrane layer. The method further includespatterning the second substrate to form a second opening therein andbonding the patterned surface of the second substrate to a surface ofthe multilayer stack. The method further includes thinning the firstsubstrate. The method further includes removing the thinned firstsubstrate and at least portions of the first and second material layersto expose opposite surfaces of the membrane layer. In some embodiments,the second opening is disposed through the second substrate. In otherembodiments, the method includes thinning the second substrate to wherethe second opening is disposed therethrough. Here, the second substratemay be thinned before or after the patterned surface thereof is bondedto the surface of the multilayer stack.

In another embodiment, a method of forming a flow cell includes forminga multilayer stack on a first substrate, the multilayer stack comprisinga membrane layer interposed between a first material layer and a secondmaterial layer, where the membrane layer features a first opening formedtherethrough. The method further includes patterning a surface of asecond substrate to form a second opening therein, bonding the patternedsurface of the second substrate to a first surface of the multilayerstack, and removing the first substrate from the multilayer stack toexpose a second surface of the multilayer stack opposite of the firstsurface. The method further includes patterning a surface of a thirdsubstrate to form a third opening therein, bonding the patterned surfaceof the third substrate to the second surface of the multilayer stack,and thinning the second substrate and the third substrate to where thesecond opening and the third openings are respectively disposedtherethrough. The method further includes removing at least portions ofthe first and second material layers to expose opposite surfaces of themembrane layer.

In another embodiment a nanopore flow cell features a glass substratehaving an opening formed therethrough and a membrane layer disposed onthe glass substrate. The membrane layer features a single nanoporedisposed therethrough. The single nanopore is located in a portion ofthe membrane layer which spans the opening formed through the glasssubstrate.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the presentdisclosure can be understood in detail, a more particular description ofthe disclosure, briefly summarized above, may be had by reference toembodiments, some of which are illustrated in the appended drawings. Itis to be noted, however, that the appended drawings illustrate onlytypical embodiments of this disclosure and are therefore not to beconsidered limiting of its scope, for the disclosure may admit to otherequally effective embodiments.

FIG. 1 is a schematic cross-sectional view of a nanopore sensor,according to one embodiment.

FIG. 2 is a graph illustrating the ionic current flow through ananoscale sized opening, such as the nanopore described in FIG. 1, as abiopolymer strand is drawn therethrough.

FIG. 3 is a flow diagram setting forth a method of forming a nanoporeflow cell, according to one embodiment.

FIGS. 4A-4I illustrate various aspects of the method set forth in FIG.3.

FIG. 4J is a schematic cross sectional view of a nanopore flow cellformed according to one embodiment of the method set forth in FIG. 3.

FIG. 4K is a schematic cross sectional view of a nanopore flow cellformed according to another embodiment of the method set forth in FIG.3.

FIG. 5 is a flow diagram setting forth a method of forming a nanoporeflow cell, according to another embodiment.

FIGS. 6A-6C illustrate various aspects of the method set forth in FIG.5.

FIG. 6D is a schematic cross sectional view of a nanopore flow cellformed according to one embodiment of the method set forth in FIG. 5.

To facilitate understanding, identical reference numerals have beenused, where possible, to designate identical elements that are common tothe figures. It is contemplated that elements and features of one aspectmay be beneficially incorporated in other aspects without furtherrecitation.

DETAILED DESCRIPTION

Embodiments of the present disclosure provide devices, e.g., nanoporeflow cells, which may be used in a solid-state nanopore sensor, andmethods of manufacturing the same. The methods described hereingenerally include forming a patterned multilayer stack on a sacrificialmonocrystalline silicon substrate before transferring the patternedmultilayer stack to a host substrate. The patterned multilayer stacktypically features a membrane layer having a nanoscale opening disposedtherethrough. The host substrate is typically formed of a dielectricglass material. Thus, the nanopore flow cells formed herein aresubstantially free of monocrystalline silicon materials. Beneficially,the glass material of the host substrate eliminates or substantiallyreduces background noise levels associated with solid-state nanoporeflow cells comprising a monocrystalline silicon substrate.

FIG. 1 is a schematic cross-sectional view of a nanopore sensor 100which may be used to sequence a biopolymer strand, according to oneembodiment. Here, the nanopore sensor 100 features a flow cell 101interposed between a first reservoir 102 and a second reservoir 103.Here, each of the first and second reservoirs 102, 103 contain anelectrically conductive fluid and a respective electrode 104, 105 whichis in communication with a voltage source 106. The voltage source 106 isused to produce an ionic current flow from the first reservoir 102 tothe second reservoir 103 through a single nanoscale sized opening, herethe nanopore 108. The nanopore 108 is disposed through a dielectricmembrane layer 109 of the flow cell 101.

Here, the ionic current flow draws a characteristically negativelycharged DNA or RNA biopolymer strand, e.g. one of the biopolymer strands107 from the first reservoir 102 through the nanopore 108 and into thesecond reservoir 103. As the biopolymer strand 107 is drawn through thenanopore 108 the monomer units thereof sequentially occlude the nanopore108 causing a change in the ionic current flow therethrough. Typically,the change in the ionic current flow corresponds to a characteristic,such as a dimension or charge, of the monomer unit simultaneouslypassing through the nanopore 108. Here, the ionic current flow andchanges in the ionic current flow are measured using an ion currentsensor, such as a pico ammeter 110.

FIG. 2 is a graph 200 illustrating the ionic current flow through ananoscale sized opening, such as the nanopore 108 described in FIG. 1,as a biopolymer strand or portion thereof, e.g., a DNA strand or RNAstrand, passes therethrough. Here, the graph 200 shows a baseline value201 where no biopolymer strand is occluding the opening and ioniccurrent flows freely therethrough. As the biopolymer strand is drawninto the nanopore, a monomer unit thereof occludes a portion of thenanopore causing the ionic current flow to change to a first value 202.As successive monomer units occlude the nanopore, i.e., as thebiopolymer strand is drawn further therethrough, the ionic current flowchanges to corresponding values 203-206 which are dependent on thepercentage of the cross-sectional area of the nanopore occluded by thebiopolymer strand. The sequential values 202-206 corresponding tomonomer units of the biopolymer strand can thus be used to determine amonomer unit sequence, e.g., a DNA or RNA base sequence, of thebiopolymer strand.

FIG. 3 is a flow diagram setting forth a method 300 of forming ananopore flow cell, according to one embodiment. FIGS. 4A-4I illustratevarious aspects of the method 300 set forth in FIG. 3.

At activity 301 the method 300 includes forming a multilayer stack on afirst substrate 401, shown in FIG. 4A. The multilayer stack features amembrane layer 403 interposed between a first material layer 402 and asecond material layer 405. The membrane layer 403 has a first opening404, e.g., a single nanopore formed there through. Typically, the firstsubstrate 401 is formed of monocrystalline silicon and has a thicknessT(1). The thickness T(1) is selected to facilitate handling andprocessing of the first substrate 401 using the same or similarequipment and methods used for processing silicon substrates in asemiconductor device manufacturing facility. In some embodiments, thefirst substrate 401 has a thickness T(1) of between about 450 μm andabout 800 μm, such as between about 600 μm and about 800 μm, forexample, between about 700 μm and about 800 μm.

Here, forming the multilayer stack includes depositing the firstmaterial layer 402 onto the first substrate 401, depositing a membranelayer 403 over the first material layer 402, and patterning the membranelayer 403 to form a first opening 404 therethrough, such as shown inFIGS. 4A-4B. In some embodiments, forming the multilayer stack furtherincludes depositing the second material layer 405 over the membranelayer 403, such as shown in FIG. 4C In some embodiments, the multilayerstack does not include the first material layer 402. In thoseembodiments, the multilayer stack includes the membrane layer 403,deposited onto the first substrate 401, and the second material layer405 deposited onto the membrane layer 403.

Typically, the first material layer 402 is formed of a dielectricmaterial, such as a silicon oxide (Si_(x)O_(y)), for example, SiO₂.Here, the first material layer 402 is deposited to a thickness T(2) ofmore than about 10 nm, such as between about 10 nm and about 500 nm,between about 10 nm and 400 nm, between about 10 nm and about 300 nm,for example between about 10 nm and about 200 nm. In other embodiments,the first material layer 402 is deposited to a thickness T(2) of morethan about 1 μm, such as more than about 2 μm, or more than about 3 μm,for example between about 4 μm and about 6 μm.

The membrane layer 403 is formed of a dielectric material which isdifferent from the dielectric material(s) used to form the first andsecond material layers 402, 405. For example, in some embodiments themembrane layer 403 is formed of a silicon nitride or silicon oxynitridematerial, such as Si_(x)N_(y) or SiO_(x)N_(y). Typically, the membranelayer 403 is deposited to a thickness T(3) of about 500 nm or less, suchas about 400 nm or less, about 300 nm or less, about 200 nm or less,about 100 nm or less, or about 50 nm or less, for example between about0.1 nm and about 100 nm or between about 1 nm and about 100 nm.

The first opening 404 is formed to extend through the membrane layer 403and to have a diameter D of less than about 100 nm, such as less thanabout 50 nm, or between about 0.1 nm and about 100 nm, for examplebetween about 1 nm and about 100 nm, or between about 0.1 nm and about50 nm. Here, the first opening 404 is formed using one or a combinationof suitable lithography and material etching patterning methods.Typically, suitable lithography methods include nanoimprint lithography,directed self-assembly, photolithography, ArF laser immersionlithography, deep UV lithography, or combinations thereof.

Here, the second material layer 405, deposited over the membrane layer403, is formed of a dielectric material which may be the same ordifferent from the dielectric material used to form the first materiallayer 402. In some embodiments, the second material layer 405 isdeposited to a thickness T(4) of between about 10 nm, such as betweenabout 10 nm and about 500 nm, between about 10 nm and 400 nm, betweenabout 10 nm and about 300 nm, for example between about 10 nm and about200 nm. Herein, the layers of the multilayer stack may be formed usingany suitable deposition method. For example, in some embodiments, thelayers of the multilayer stack are deposited using one, or acombination, of chemical vapor deposition (CVD) or physical vapordeposition (PVD) methods.

At activity 302 the method 300 includes patterning a surface of a secondsubstrate 407 to form an opening therein, here the second opening 409shown in FIGS. 4D-4E. Typically, the second substrate 407 is formed ofdielectric material having a thickness T(5) selected to facilitatehandling and processing of the second substrate 407 using the same orsimilar equipment used for processing silicon substrates in asemiconductor device manufacturing facility. For example, in someembodiments, the second substrate 407 has a thickness T(5) of betweenabout 450 μm and about 800 μm, such as between about 600 μm and about800 μm, for example, between about 700 μm and about 800 μm. In otherembodiments, the second substrate 407 has a thickness of about 400 μm orless, such as about 300 μm or less, for example, about 300 μm.

Here, the second substrate 407 is formed, for example, of anon-crystalline amorphous solid, i.e., glass, such as a transparentsilica-based glass material, for example, a fused silica, i.e., anamorphous quartz material, or a borosilicate glass material. In someembodiments, the second substrate 407 has an opaque material layer 408,for example, an amorphous silicon layer deposited on a backside surfacethereof. The backside surface of the second substrate 407 is opposite ofthe surface to be patterned, here the front side surface into which thesecond opening 409 is formed. When used, the opaque material layer 408typically has a thickness T(6) of about 20 nm or more, for example,about 100 nm or more. The opaque material layer 408 facilitates thedetection of an otherwise optically transparent substrate, according tosome embodiments, by optical sensors of conventional semiconductordevice manufacturing equipment.

Here, the second opening 409 is formed to extend from a surface of thesecond substrate 407, here the patterned surface, to a depth H ofbetween about 100 μm or more and less than the thickness T(5) of thesecond substrate 407. For example, in some embodiments, the depth H ofthe second opening 409 extends between about 100 μm and about 600 μm, orbetween about 200 μm and about 400 μm, from the front side surface ofthe second substrate 407. In some other embodiments, such as inembodiments where the thickness of the second substrate 407 is less thanabout 400 μm the second opening 409 is formed to extend through thethickness of thereof.

Here, the second opening 409 is formed to have a width W(1) of betweenabout 1 μm and about 20 μm, such as between about 1 μm and about 15 μm,between about 5 μm and about 15 μm, or between about 5 μm and about 10μm. The second opening 409 may be formed using any suitable combinationof photolithography and material etching patterning methods.

At activity 303 the method 300 includes bonding the patterned surface ofthe second substrate 407 to an exposed surface of the multilayer stackdisposed on the first substrate 401, such as shown in FIGS. 4F-4G.Typically, the patterned surface of the second substrate 407 and theexposed surface of the multilayer stack are bonded together using asuitable direct bonding method. Direct bonding describes methods ofjoining two substrate surfaces at an atomic level, e.g., throughchemical bonds between the substrates, without the use of intermediatelayers, such as conductive adhesive layers, solders, etc., interposedtherebetween. In one example, a suitable direct bonding method includesplasma activating one or both of the surfaces to be bonded of thesubstrates 401, 407, contacting the surfaces to be bonded, applying acompressive bonding force to the contacted substrates to form acomposite substrate, and annealing the composite substrate.

Herein, bonding the patterned surface of the second substrate 407 to theexposed surface of the multilayer stack includes aligning the secondopening 409 407 with the first opening 404. When the first and secondsubstrates 401, 407 are properly aligned, the first opening 404 and thesecond opening 409 in the resulting nanopore flow cell will be in fluidcommunication, e.g., a portion of the membrane layer 403 having thefirst opening 404 formed therethrough will span the second opening 409formed in the second substrate 407.

At activity 304 the method 300 includes thinning the first substrate401. Thinning the first substrate 401 includes any one or combination ofgrinding, lapping, chemical mechanical planarization (CMP), etching, orcleaving methods which may be used to achieve a desired thickness T(7)(shown in FIG. 4H). In embodiments where thinning the first substrate401 comprises a cleaving method, a surface of the first substrate 401 istypically implanted with one or a combination of hydrogen or helium ionsto a depth of about 100 nm before forming the multilayer stack thereon.The implant process desirably introduces a layer of damage, e.g.,microbubbles, into the first substrate 401 to facilitate cleaving of thefirst substrate 401 along the damaged layer. Typically, the firstsubstrate 401 is thinned to a thickness T(7) of less than about 100 μm,such as less than about 50 μm, less than about 10 μm, or for example,less than about 1 μm. In some embodiments, the first substrate 401 isthinned to a thickness T(7) less than about 500 nm, such as less thanabout 200 nm, for example, about 100 nm or less.

At activity 305, the method 300 includes thinning the second substrate407 using any one or combination of grinding, lapping, CMP, or etchingmethods to achieve a desired thickness T(8) (shown in FIG. 4I). Here,the second substrate 407 is thinned until the second opening 409 isdisposed therethrough, i.e., the thickness T(8) is the same or less thanthe depth H of the second opening 409 formed in the patterned surface atactivity 302. For example, in some embodiments, the thickness T(8) ofthe thinned second substrate 407 is less than about 700 μm, such as lessthan about 600 μm, less than about 500 μm, for example, less than about400 μm or between about 100 μm and about 700 μm, such as between about200 μm and about 500 μm. In some embodiments, the second substrate 407is thinned before the patterned surface thereof is bonded to the surfaceof the multilayer stack.

At activity 306, the method 300 includes removing the thinned firstsubstrate 401 and at least portions of the first and second materiallayers 402, 405 to expose opposite surfaces of the membrane layer 403spanning the second opening 409, such as shown in FIG. 4J or 4K. In someembodiments, removing the thinned first substrate 401 and at leastportions of the first and second material layers 402, 405 includesexposure thereof to a wet or aqueous etchant, such as KOH or acombination of KOH and HF.

In some embodiments, such as shown in FIG. 4J, all or substantially allof the first material layer 402 is removed from the surface of themembrane layer 403 disposed distal from the second substrate 407. Inother embodiments, such as shown in FIG. 4K, a third opening 412 isformed in the first material layer 402 and a surface of the membranelayer 403 is exposed therethrough. The third opening 412 may be formedusing any suitable combination of photolithography and material etchingpatterning methods, e.g., a plasma-assisted etching or a wet etching(aqueous solution) process.

FIG. 4J is a schematic cross-sectional view of a flow cell 410, formedaccording to the method set forth in FIG. 3, which may be used in placeof the flow cell 101 described in FIG. 1. Here, the flow cell 410includes the second substrate 407, having the thickness T(8), and thesecond material layer 405, having the thickness T(4), disposed on thesecond substrate 407. The second opening 409, having the width W(1), isdisposed through the second substrate 407, and further through thesecond material layer 405. The membrane layer 403, having the thicknessT(3) and the first opening 404 disposed therethrough, is disposed on thesecond material layer 405 and spans the second opening 409. Here, thefirst opening 404 is in fluid communication with the second opening 409.

FIG. 4K is a schematic cross-sectional view of a nanopore flow cell 411,formed according to the method set forth in FIG. 3, which may be used inplace of the flow cell 101 described in FIG. 1. Here, the flow cell 411is substantially the same as the flow cell 410 described in FIG. 4J andfurther includes the first material layer 402 disposed on the membranelayer 403, the first material layer 402 having an opening, here thethird opening 412, disposed therethrough. Here, a thickness T(9) of thefirst material layer 402 is between about 1 μm and about 5 μm. In someembodiments, a width of the third opening 412 is the same as the widthW(1) of the second opening 409. In other embodiments, the width of thethird opening 412 is less than or more than the width W(1) of the secondopening 409. Here, the third opening 412 is in fluid communication withthe second opening 409 and the first opening 404 is disposedtherebetween.

FIG. 5 is a flow diagram setting forth a method of forming a flow cell,according to another embodiment. FIGS. 6A-6C illustrate various aspectsof the method set forth in FIG. 5 in addition to the aspects illustratedin FIGS. 4A-4H. Here, activities 501-502 of the method 500 are the sameas activities 301-302 of the method 300 set forth in FIG. 3, illustratedin FIGS. 4A-4E, and described above.

Activity 503 of the method 500 includes bonding the patterned surface ofthe second substrate 407 to an exposed surface of the multilayer stack,here a first surface, such as described in activity 303 of the method300 set forth in FIG. 3 and illustrated in FIG. 4F.

At activity 504, the method 500 includes removing the first substrate401 from the multilayer stack to expose a second surface of themultilayer stack. Here, the second surface of the multilayer stack isopposite of the first surface and is disposed proximate to the firstsubstrate 401 before the first substrate 401 is removed therefrom.Removing the first substrate 401 from the multilayer stack may includeany one or combination of grinding, lapping, chemical mechanicalplanarization (CMP), etching, or cleaving methods described in activity304 of the method 300 set forth in FIG. 3.

At activity 505, the method 500 includes patterning a surface of a thirdsubstrate, such as the third substrate 607 shown in FIG. 6A, to form anopening therein, here the third opening 609. In some embodiments, thethird substrate 607 is formed of the same dielectric material used toform the second substrate 407 and has the same or substantially the samethickness T(5). In some embodiments, the third substrate 607 ispatterned using the methods of patterning the second substrate 407described in activity 302 of the method 300. In some embodiments, theopening 609 is formed to have the same width W(1) and depth H as thesecond opening 409 in the second substrate 407. In some embodiments, thethird substrate 607 includes an opaque material layer 608 deposited on abackside surface thereof. In some embodiments, the opaque material layer608 is formed of the same material and has the same thickness T(6) asthe opaque material layer 408 disposed on the second substrate 407. Inother embodiments, the third substrate 607 is formed of a dielectricmaterial which is different than the dielectric material of the secondsubstrate 407, patterned using a different method than the methods setforth in activity 302 of the method 300, and/or the opening is formed tohave a different width and depth than the width W(1) and depth H of thesecond opening 409.

At activity 506, the method 500 includes bonding the patterned surfaceof the third substrate 607 to the second surface of the multilayer stackusing a suitable direct bonding method. A suitable direct bonding methodis described at activity 303 of the method 300 set forth in FIG. 3.Herein, bonding the patterned surface of the third substrate 607 to thesecond surface of the multilayer stack includes aligning the thirdopening 609 formed in the third substrate 607 with the first opening 404formed in the membrane layer 403, such as shown in FIGS. 6A-6B.

At activity 507, the method 500 includes thinning the second and thirdsubstrates 407, 607 to thickness T(8) where the second and thirdopenings 409, 609 are respectively disposed therethrough. Typically,thinning the second and third substrates 407, 607 includes any one orcombination of grinding, lapping, CMP, or etching methods to achieve thedesired thickness T(8), shown in FIG. 6C, which may be the same ordifferent for each of the second and third substrates 407, 607respectively.

At activity 508, the method 500 includes removing at least portions ofthe first and second material layers 402, 405 to expose oppositesurfaces of the membrane layer 403, such as shown in FIG. 6D. In someembodiments, removing the at least portions of the first and secondmaterial layers 402, 405 comprises exposure thereof to an etchant, suchas KOH or a combination of KOH and HF.

FIG. 6D is a schematic cross-sectional view of a flow cell 610 formedusing the method set forth in FIG. 5, which may be used in place of theflow cell 101 described in FIG. 1. Here, the flow cell 610 issubstantially the same as the flow cell 410 described in FIG. 4J andfurther includes the first material layer 402, having the thicknessT(4), disposed on the membrane layer 403, and the third substrate 607disposed on the first material layer 402. Here, the third substrate 607has a thickness that is the same and the thickness T(8) of the secondsubstrate 407. In other embodiments, the thicknesses of the second andthird substrates 407, 607 are different. The third opening 609 isdisposed through the third substrate 607 and further through the firstmaterial layer 402. A width of the third opening 609 is the same as thewidth W(1) of the second opening 409. In other embodiments, the width ofthe third opening 609 is less than or more than the width W(1) of thesecond opening 409. In embodiments herein, the third opening 609 is influid communication with the first opening 404 and the second opening409.

While the foregoing is directed to embodiments of the presentdisclosure, other and further embodiments of the disclosure may bedevised without departing from the basic scope thereof, and the scopethereof is determined by the claims that follow.

1. A flow cell device, comprising: a glass substrate; and a membranelayer disposed on the glass substrate, the membrane layer having ananopore disposed therethrough, and the nanopore is located in a portionof the membrane layer which spans an opening formed through the glasssubstrate.
 2. The flow cell device of claim 1, wherein the glasssubstrate is formed of fused silica, borosilicate, or a combinationthereof.
 3. The flow cell device of claim 2, wherein the nanopore has adiameter of about 100 nm or less.
 4. The flow cell device of claim 3,wherein a thickness of the membrane layer is less than about 100 nm. 5.The flow cell device of claim 1, wherein the membrane layer isinterposed between a first dielectric layer and a second dielectriclayer to form a multilayer stack.
 6. The flow cell device of claim 5,wherein the first and second dielectric layers are formed of siliconoxide, and the membrane layer is formed of silicon nitride or siliconoxynitride.
 7. The flow cell device of claim 5, wherein respectiveopenings formed through each of the first and second dielectric layersexpose opposite surfaces of the membrane layer.
 8. The flow cell deviceof claim 5, wherein the second dielectric layer is disposed on the glasssubstrate, the membrane layer is in contact with the second dielectriclayer, and the first dielectric layer is in contact with the membranelayer.
 9. The flow cell device of claim 8, wherein the glass substrateis a first substrate and the flow cell device further comprises a secondsubstrate disposed on and in contact with the first dielectric layer,wherein an opening formed through the second substrate is aligned withthe opening formed through the first dielectric layer, and the secondsubstrate is formed of fused silica, borosilicate, or a combinationthereof.
 10. A nanopore sensor for biopolymer strand sequencing,comprising: a flow cell interposed between a first reservoir and asecond reservoir, the flow cell comprising a glass substrate and amembrane layer disposed on the glass substrate, the membrane layerhaving a nanopore formed therethrough, and the nanopore is located in aportion of the membrane layer which spans an opening formed through theglass substrate.
 11. The nanopore sensor of claim 10, wherein the glasssubstrate is formed of fused silica, borosilicate, or a combinationthereof.
 12. The nanopore sensor of claim 10, wherein the nanopore has adiameter of about 100 nm or less.
 13. The nanopore sensor of claim 10,wherein the first and second reservoirs each contain an electricallyconductive fluid and a respective electrode that is in communicationwith a voltage source.
 14. The nanopore sensor of claim 13, wherein thevoltage source is configured to produce an ionic current flow throughthe nanopore.
 15. The nanopore sensor of claim 14, wherein the membranelayer is interposed between a first dielectric layer and a seconddielectric layer to form a multilayer stack.
 16. The nanopore sensor ofclaim 15, wherein the second dielectric layer is disposed on the glasssubstrate, the membrane layer is disposed on and in contact with thesecond dielectric layer, and the first dielectric layer is disposed onand in contact with the membrane layer.
 17. A method of sequencing abiopolymer strand using a nanopore sensor, comprising: generating acurrent between a first reservoir and a second reservoir to draw thebiopolymer strand through a nanopore of a flow cell interposed betweenthe first reservoir and the second reservoir, wherein the flow cellcomprises a membrane layer disposed on a glass substrate, the nanoporeis formed through a portion of the membrane layer that spans an openingformed through the glass substrate, and the biopolymer strand comprisesa plurality of monomer units that sequentially occlude the nanopore asthe biopolymer strand is drawn therethrough; and determining, based onchanges in the current as the biopolymer strand is drawn through thenanopore, a monomer unit sequence of the biopolymer strand, whereinchanges in the current correspond to differences in one or morecharacteristics of each of the monomer units sequentially occluding thenanopore.
 18. The method of claim 17, wherein the glass substrate isformed of fused silica, borosilicate, or a combination thereof.
 19. Themethod of claim 17, wherein each of the first and second reservoirscontain an electrically conductive fluid and an electrode, each of theelectrodes is electrically coupled to a voltage source, and a voltagefrom the voltage source is used to generate the current.
 20. The methodof claim 17, wherein the membrane layer is interposed between a firstdielectric layer and a second dielectric layer to form a multilayerstack, and respective openings formed through each of the first andsecond dielectric layers expose opposite surfaces of the membrane layer.